The CRISPR system is a revolutionary genome editing technology that can efficiently modify target genes in mammalian cells1. It targets a short stretch of DNA via the hybridization of a complementary guide RNA (gRNA) and binding of a CRISPR nuclease, such as CAS9, that recognizes a protospacer adjacent motif (PAM) in the target gene1. Preclinical studies have shown that the CRISPR system provides unprecedented opportunities for treating a variety of genetic diseases and infectious diseases2-6.
Although the CRISPR system can have good targeting efficiency, gRNAs can also hybridize to DNA sequences containing base mismatches. Consequently, the CRISPR system can have off-target activities, causing gene mutations, deletions, inversions or translocations, which may lead to tumorigenic or other deleterious events7-10. Therefore, one of the major challenges for in vivo clinical applications of genome editing is to selectively activate the CRISPR system in the desired tissue or organ in order to maximize therapeutic efficacy and reduce genotoxicity.
To improve the specificity of CRISPR systems, many tools have been developed for identifying potential gRNA off-target sites11, and CAS9 and other CAS nucleases have been designed with controllable nuclease activities12, 13. For example, CAS9 proteins have been fragmented into nonfunctional units, which can dimerize to form active nucleases upon blue light radiation13. CAS9 can also be delivered as inducible transgenes that can only be translated in the presence of a chemical cue, e.g. doxycycline12. However, for in vivo applications, optical signals cannot penetrate deeply into the body owing to the strong absorption and scattering of light by biological tissues14, and the chemically-regulated CAS9 expression relies on the biodistribution of transgenes.
An alternative approach to controlling in vivo genome editing is through targeted delivery of the CRISPR system. In particular, the viral vectors with tissue tropism, e.g., the adeno-associated viral vector (AAV)15, are being explored for tissue-specific genome editing in vivo3, 10, 16. However, most viral vectors for in vivo gene delivery are derived from viruses originating from human or other mammals. It is difficult to control the systemic dissemination and replication of these viral vectors used for in vivo genome editing, which increases the risk of genotoxicity4, 17. Furthermore, vectors derived from small viruses, such as AAV and LV, have packaging limits, thus limiting the size of the genetic material that can be introduced. In addition, many viruses are targeted by the complement cascade for inactivation, thus limiting their efficiency.
The Baculoviridae is a family of viruses that infect insects and are very large—their circular double-stranded genome ranging from 80-180 kbp. Because of this large size, they have great potential as a vector, and many such vectors are in use since Dr. Max Summers developed the first baculovirus expression vector system.
The large size of BVs allows an extraordinary DNA packing capacity compared to most other viruses, thus enabling the integration of multiple gene expression cassettes into a single viral vector23. Although the baculovirus and their vectors lack the ability to replicate in mammalian cells, they can transduce mammalian cells with high efficiency and low cytotoxicity, providing a robust and transient gene expression22-25. However, there have been very limited in vivo applications of BVs because of their inactivation by the complement cascade in the serum24, 26.
The complement system represents a first-line host defense of the innate immune system designed to eliminate foreign elements, such as insect viruses. It has been well established that BV administrated intravenously can circulate throughout the body, and the complementary factor C3 in the blood will bind to circulating BV and initiate molecular events that eventually lead to BV inactivation (FIG. 1)26. Indeed, triggering of the complement cascade is a major cause for the inactivation of a variety of currently used gene delivery vectors and contributes to inefficient gene transfer rates after in vivo application.
Thus, what is needed in the art are better nucleic acid delivery methods, products, and systems that solve or at least mitigate one or more of the above limitations. The ideal gene delivery mechanism would allow targeted delivery of nucleic acid, and avoid off-target effects.